ES2301697T3 - MATTERS OF MODIFIED PROTEIN WITH GROWTH FACTOR FOR TISSUE ENGINEERING. - Google Patents

Abstract

Proteins are incorporated into protein or polysaccharide matrices for use in tissue repair, regeneration and/or remodeling and/or drug delivery. The proteins can be incorporated so that they are released by degradation of the matrix, by enzymatic action and/or diffusion. As demonstrated by the examples, one method is to bind heparin to the matrix by either covalent or non-covalent methods, to form a heparin-matrix. The heparin then non-covalently binds heparin-binding growth factors to the protein matrix. Alternatively, a fusion protein can be constructed which contains a crosslinking region such as a factor XIIIa substrate and the native protein sequence. Incorporation of degradable linkages between the matrix and the bioactive factors can be particularly useful when long-term drug delivery is desired, for example in the case of nerve regeneration, where it is desirable to vary the rate of drug release spatially as a function of regeneration, e.g. rapidly near the living tissue interface and more slowly farther into the injury zone. Additional benefits include the lower total drug dose within the delivery system, and spatial regulation of release which permits a greater percentage of the drug to be released at the time of greatest cellular activity.

Description

Modified protein matrices with factor tissue engineering growth.

Field of the Invention

The invention relates to proteins of fusion or peptides containing PTH and an amino acid sequence which allows union interactions with matrices and with the use of fusion proteins or peptides in the repair and regeneration of tissues, and in the controlled release of PTH.

Background of the invention

Parathyroid hormone (PTH) is an 84 amino acid peptide that is made and secreted by the parathyroid gland. This hormone plays a primary role in controlling serum calcium levels through its action on various tissues, including bones. Studies in humans with various forms of PTH have shown an anabolic effect on bones, which makes them interesting for the treatment of osteoporosis and related bone disorders (U.S. Patent No. 5,747,456 to Chorev, et al . And WO 10/10596 from Eli Lilly & Co.). Parathyroid hormone acts on cells by binding to a cell surface receptor. It is known that this receptor is found on osteoblasts, the cells that are responsible for the formation of new bones.

It has been reported that the N-terminal 34 amino acid domain of the human hormone is biologically equivalent to the full length hormone. PTH 1-34 and its mode of action were first reported in U.S. Patent No. 4,086,196. Because the research has been conducted on PTH 1-34 and other truncated versions of the natural human PTH form, such as PTH1-25, PTH1-31 and PTH1-38 (see, for example, Rixon RH, et al ., J Bone Miner. Res. , 9 (8): 1179-89 (August 1994).

The mechanism by which PTH influences. Bone remodeling is complicated, which has led to conflicting results and subsequently to a significant number of studies on the exact mechanism involved. Has been shown that if PTH is administered systemically on an ongoing basis, bone density will decrease. On the contrary, it has been reported that if the same molecule is administered in a pulsatile manner, the bone density (see, for example, WO 99/31137 by Eli Lilly & Co.). This apparent contradiction can be explained by the mechanism by which PTH modulates bone remodeling and subsequently the visible parameter of bone density. Inside of the mature bone, the PTH receptor has only shown to be present on the surface of the osteoblast lineage cells, but not about osteoclasts. The role that PTH plays in bone remodeling is directed through osteoblasts in opposition with osteoclasts. However, the cells in different stages of the osteoblast lineage responds so different when they join the PTH. Therefore, the dramatic differences that are observed when PTH is administered using different methods can be taken into account for the understanding of the different effects that the same molecule has on the different cells within the osteoblast lineage.

When PTH binds to a hemocytoblast mesenchymal, the cell is induced to differentiate into a proteoblast. Thus, when adding PTH to the system, there is an increase in the population of proteoblasts. However, you are Proteoblastic cells also have the PTH receptor, and the subsequent binding of PTH to the receptor on these cells leads to A different answer. When PTH binds to the proteoblast, this results in two separate consequences that lead to bone resorption, first, inhibits differentiation additional proteoblasts in osteoblasts. Secondly, Increases the secretion of Interleukin 6 (IL-6) from proteoblasts. IL-6 both inhibits proteoblast differentiation, as well as increases differentiation of proteoclasts inside osteoclasts. This dual response of the cells within the lineage of osteoblasts is that it provides the complex reaction between the bone remodeling and exposure to PTH. If the PTH is dosed periodically for short periods of time, then the mesenchymal hemocytoblasts are induced to differentiate in osteoblasts Short dosing periods then avoid that newly formed proteoblasts produce IL-6, preventing the activation of osteoclasts. Therefore, during dosing intervals, these newly formed preosteoblasts can be differentiated additionally in osteoblasts, resulting in formation that is. However, if a constant dose of PTH is applied, then the preosteoblasts will have the opportunity to start the IL-6 production, thus activating osteoclasts and inhibiting them, leading to the opposite effect: resorption that is.

For tissue repair or regeneration, the cells must migrate into a wounded mass, proliferate, express the matrix components or form the extracellular matrix, and constitute a final tissue conformation. In this answer Morphogenetic often must involve multiple populations cellular, frequently including vascular cells and nervous For this to happen, it has been shown that the matrices they improve a lot and it has been found that in some cases they are essential. Procedures have been developed to develop matrices from natural or synthetic origins or a mix of both. The growing matrices of natural cells undergo remodeling through cellular influence, all based on proteolysis, for example, by plasmin (fibrin degrading) and matrix metalloproteinazas (degrading collagen, elastin, etc.) This degradation is quite localized and is presents only at the time of direct contact with the migrant cell In addition, the supply of signaling proteins  specific cell, such as for example the factors of growth, is rigorously controlled. In the natural model, no growing matrices of macroporous cells are used, in instead of microporous matrices cells can degrade, locally and at the time of demand, as the cells They migrate into the matrix. Due to issues that relate with immunogenicity, expensive production, availability limited, variability and purification of lots, have been developed matrices based on synthetic precursor molecules, such as for example, modified polyethylene glycol in and / or on the body.

While a lot of work has been done on the study of the systemic effects of PTH, research does not has explored the local or topical administration of PTH. Half that PTH has a direct anabolic effect on the lineage Osteoblast cell, must have significant potential to cure bone defects in addition to influencing bone density if It is presented properly within a defective site. One time that the defect has been filled with preosteoblasts, if the signal from PTH is canceled, the newly formed preosteoblasts are then they can differentiate into osteoblasts and start the conversion of the wounded mass, first in wounded bone tissue and then in a mature bone structure.

Therefore, an objective of the present invention is to provide a PTH in a way that can be attached to a matrix for repair, regeneration and remodeling of tissues.

An additional objective is to present a PTH in a form suitable for topical or local administration to a patient to cure bone defects.

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Summary of the invention

The peptides of fusion containing a parathyroid hormone (PTH) in a domain and a substrate domain capable of covalently crosslinking to a matrix in another domain and matrices, and the computers it contains these fusion proteins or peptides. Fusion proteins are covalently bind to natural or synthetic materials to form matrices, can be used to cure bone defects. Optionally, all components for matrix formation they are applied to a bone defect and the matrix is formed at the site of application. The fusion peptide can be incorporated into the matrix such that the fusion peptide as a whole or only the respective PTH sequence of the first domain is released by degradation of the matrix, by enzymatic and / or hydrolytic action. Also, the fusion peptide may contain a degradable bond. between the first and second domain that contains cleavage sites Hydrolytic or enzymatic. In particular, the fusion peptide contains the PTH in a domain, a substrate domain capable of being covalently crosslinked to a matrix in a second domain, and a degradation site between the first and second domain. From Preferably, PTH is human PTH, although a PTH from other sources, such as, for example, PTH from bovine. PTH can be PTH 1-84 (natural), PTH 1-38, PTH 1-34, PTH 1-31, or PTH 1-25, or any modified or allelic versions of PTH that exhibit properties, is that is, bone formation, similar to the previous one. The site of degradation allows supply speed to vary to different locations within the matrix depending on the cellular activity in that location and / or within the matrix. Additional benefits include the lower total dose of drugs within the supply system, and the spatial regulation of the release that allows a higher percentage of the drug that will be released at the time of increased cellular activity.

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Brief description of the drawings or figures

Figure 1 is a chromatogram for detection by fluorescence of fibrin gels containing peptide degraded with plasmin and free peptide. It shows the size exclusion chromatography of a fibrin gel degraded with the peptide α2 {PI_ {1-7} -ATIII_ {121-134} incorporated (-), with the same free peptide added to the gel degraded fibrin containing the incorporated peptide (\ cdot \ cdot \ cdot), and the free peptide alone (- -). The residue of N-terminal leucine was danced (abbreviated dL). He free peptide eluted at higher times, corresponding to a weight lower molecular, than did the peptide incorporated into the fibrin gel during coagulation, demonstrating binding fibrin covalent degrades and thus incorporation covalent via the action of Factor XIIIa activity.

Figure 2 is a graph of the incorporation of dLNQEQVSPLRGD (SEQ ID NO: 1) in fibrin gels with the Exogenous factor XIII added. When 1 U / mL was added, the level of incorporation increased so that more than 25 moles of peptide / moles of fibrinogen.

Figure 3 is a graph of the incorporation of the dLNQEQVSPLRGD two domain peptide (SEQ ID NO: 1), in rubber undiluted fibrin. Three separate reagent kits were tested and in each case a high level of incorporation could be observed, reaching 25 moles of fibrinogen peptide / moles. The concentration of exogenous peptide required for incorporation maximum was at least 5 mM, possibly due to limitations of diffusion within the matrix of dense fibrin that is created. The level of incorporation was very consistent, with each team of reagents that provide a similar incorporation profile.

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Detailed description of the invention

Products and methods for the repair, regeneration or remodeling of hard tissue are described herein, in particular for bone growth, using natural and synthetic matrices having the PTH that can be distributed incorporated herein. Natural matrices are biocompatible and biodegradable and can be formed in vitro or in vivo , at the time of implantation. PTH can be incorporated into the matrices and maintain its total bioactivity. PTH can be incorporated so that it can be distributed, using techniques that provide control over the form and timing and to what degree PTH is released, so that the matrix can be used to repair tissues directly or indirectly, using the matrix as a controlled release vehicle.

Definitions

"Biomaterial", in the sense in which used herein, refers to a material intended for interconnect with biological systems to evaluate, treat, increase, or replace any tissue, organ or function of the body depending on the material either permanently or temporarily. The terms "biomaterial" and "matrix" are used interchangeably in the present and mean a polymeric network degraded that, depending on the nature of the matrix, can increase with water but not dissolve in water, that is, form a hydrogel that remains in the body for a certain period of time fulfilling certain support functions for tissue  hard traumatized or defective.

"PTH fusion peptide" in the sense  in which it is generally used herein, refers to a peptide containing at least a first and a second domain. A domain contains a PTH (natural or truncated forms, in particular PTH 1-34), and the other domain contains a Substrate domain that will be degraded to an array. A site of enzymatic or hydrolytic degradation may also be present between the first and second domain.

"Strong nucleophile", in the sense in the which is generally used herein, refers to a molecule that is able to donate a pair of electrons to a electrophile in a polar bond forming reaction. From Preferably, the strong nucleophile is more nucleophilic than water at a physiological pH Examples of strong nucleophiles are thiols and amines

"Conjugated unsaturated bond", in the sense in which it is generally used herein, it refers to the alternation of multiple links carbon-carbon, carbon-heteroatom or heteroatom-heteroatom with individual bonds, or the binding of a functional group to a macromolecule, such as by example, a synthetic polymer or a protein. These links can experience addition reactions.

"Conjugated unsaturated group", in the sense  in which it is generally used herein, refers to a molecule or a region of a molecule, which contains an alternation of carbon-carbon bonds, carbon-heteroatom or heteroatom-heteroatom, with individual bonds, that have a multiple bond that can experience reactions of addition. Examples of conjugated unsaturated groups include enunciative way: vinyl sulfones, acrylates, acrylamides, quinones, and vinyl pyridiniums, for example, 2- or 4-vinylpyridinium and itaconatos.

"Synthetic precursor molecules", in the sense in which it is generally used herein, it refers to molecules that do not exist in nature.

"Precursor components or polymers that are present in nature ", in the sense in which it is used generally herein, refers to molecules that could be Find in nature.

"Functionalize", in the sense in which  generally used herein, refers to the modification of a molecule in a way that results in the union of a functional group or entity. For example, a molecule can have a functional group by introducing a molecule that turn the molecule into a strong nucleophile unsaturation conjugated Preferably, a molecule, for example PEG, is provides with a functional group to convert to a thiol, amine, acrylate, or quinone. Proteins, in particular, too they can integrate functional groups effectively through partial or complete reduction of disulfide bonds to create thiols free.

"Functionality", in the sense in which  generally used herein refers to the number of sites Reagents in a molecule.

"Functionality of the points of branching ", in the sense in which it is generally used herein, it refers to the number of branches that are extend from a point in the molecule.

"Accession site or binding site cellular ", in the sense in which it is generally used in this refers to a sequence of peptides to which binds a molecule, for example, an adhesion stimulator receptor on the surface of a cell. Examples of sites from Adhesion include the following: the RGD sequence from fibronectin, and the YIGSR sequence (SEQ ID NO: 2) from laminin. Preferably, the adhesion sites are incorporated into the biomaterial by including a substrate domain that I can degrade with a matrix.

"Biological activity", in the sense in the which is generally used herein refers to events functional caused by a protein of interest. In some modalities, this includes the events analyzed when measuring interactions of a polypeptide with another polypeptide. Too include analyzing the effect that the protein of interest has on growth, differentiation, death, migration, adhesion, cellular interactions with other proteins, activity Enzymatic, protein phosphorylation or dephosphorylation, transcription or translation

"Sensitive biological molecule" in the sense  in which it is generally used herein, refers to a molecule found in a cell, or in a body, or that is can use as a therapeutic agent for a cell or a body, which can react with other molecules in its presence. Examples of sensitive biological molecules include enunciative: peptides, proteins, nucleic acids and drugs. Be they can produce biomaterials in the presence of materials Biologically sensitive, without negatively affecting materials Biologically sensitive

"Regenerate", in the sense in which generally used in the present, means to grow again a portion or all of something, such as by example, hard tissue, particularly bones.

"Multifunctional", in the sense in which It is generally used herein, it refers to more than one electrophilic and / or nucleophilic functional group per molecule (it is say, monomer, oligo and polymer).

"Self-selective reaction" in the sense that it is generally used herein, means that the first precursor component of a composition reacts much faster with the second precursor component of the composition and vice versa than with other compounds present in a mixture or reaction site. In the sense in which it used herein, the nucleophile preferably binds to a electrophile and an electrophile preferably binds to a nucleophile strong, instead of other biological compounds.

"Degradation", in the sense in which generally used herein, means the formation of covalent bonds However, you can also refer to Link formation non-covalent, such as, for example, ionic bonds, or combinations of covalent bonds and not covalent

"Polymeric network", in the sense in which is generally used herein, means the product of a process in which virtually all monomers, oligo or polymers are joined by intermolecular covalent bonds to through its functional groups available to produce a large molecule

"Physiological", in the sense in which generally used herein, means the conditions as can be found in living vertebrates. In particular, physiological conditions, refers to the conditions in the body human such as for example, pH temperature, etc. Temperatures physiological means in particular a temperature variation between 35 ° C to 42 ° C, preferably approximately 37 ° C.

"Density of degradation", in the sense in  the one generally used herein refers to the weight molecular average between two degradations (Me) of the molecules respective.

"Equivalent weight", in the sense in which  generally used herein refers to mmol of the group Functional / g of the substance.

"Increase in volume", in the sense in the which is generally used herein refers to the increase in volume and mass by the proportion of water by the biomaterial The terms "water absorption" and "increased volume "are used interchangeably throughout this application.

"State of equilibrium", in the sense in the which is generally used herein means the state in which a hydrogel does not experience increase or decrease in mass when stored under constant conditions in water.

I. Matrices and PTH A. Matrix materials

The matrix is formed by degrading ionic, covalently, or by combinations thereof, the precursor molecules for a polymeric network or when increasing from volume one or more polymeric materials, that is, the matrices, to form a polymeric network that has sufficient separation inter-polymer to allow growth interior or migration in the matrix of the cells.

In one embodiment, the matrix is formed of proteins, preferably proteins naturally present in the patient, within which the matrix will be implanted. A protein Particularly preferred matrix is fibrin, although it is also they can use the elaborated matrices of other proteins, such such as collagen or gelatin. Can also be used polysaccharides and glycoproteins to form the matrix. It is also possible to use synthetic polymers that can be degraded by phonic or covalent agglutination.

Fibrin matrices

Fibrin is a natural material that has been reported for various biomedical applications. Fibrin has been described as a material for internal cell growth matrices in U.S. Patent No. 6,331,422 to Hubbell et al . Fibrin gels have been used as sealants due to their ability to bind to many tissues and their natural function is wound healing. Some specific applications include the use as a sealant for vascular graft attachment, cardiac valve attachment, fracture bone placement and tendon repair (Sierra, DH, Journal of Biomaterials Applications , 7 : 309-352 (1993) ). Additionally, these gels have been used as devices for drug delivery, and for neuronal regeneration (Williams, et al ., Journal of Comparative Neurobiology , 264 : 284-290 (1987)). Although fibrin provides a solid support for tissue regeneration and internal cell growth, there are few active sequences in the monomer that directly improve these processes.

The process by which the fibrinogen polymerizes inside the fibrin has also been characterized. Initially, a protease cleaves the dimeric fibrinogen molecule at two symmetric sites. There are several possible proteases that can cleave fibrinogen, including thrombin, reptilase, and protease III, and each serves the protein at a different site (Francis, et al ., Blood Cells , 19 : 291-307, 1993). Once the fibrinogen is cleaved, a self-polymerization step occurs in which the fibrinogen monomers come together and form a non-covalently degraded polymer gel (Sierra, 1993). This self-assembly occurs because the binding sites are exposed after protease cleavage occurs. Once exposed, these binding sites in the center of the molecule can bind to other sites of the fibrinogen chains, which are present at the ends of the peptide chains (Stryer, L. In Biochemistry , WH Freeman & Company , NY, 1975). In this way, a polymeric network is formed. Factor XIIIa, a transglutaminase activated from Factor XIII by thrombin proteolysis, can then covalently degrade the polymer network. Other transglutaminases exist and may also be involved in covalent degradation and graft formation with the fibrin network.

Once a degraded fibrin gel is formed, subsequent degradation is rigorously controlled. One of the key molecules to control fibrin degradation is the α2-plasmin inhibitor (Aoki, N., Progress in Cardiovascular Disease , 21 : 267-286, 1979). This molecule acts by degrading the fibrin α chain through the action of Factor XIIIa (Sakata, et al ., Journal of Clinical Investigation , 65 : 290-297, 1980). By binding itself to the gel, a high concentration of the inhibitor can be located with the gel. The inhibitor then acts by preventing the binding of plasminogen with fibrin (Aoki, et al .; Thrombosis and Haemostasis , 39 : 22-31, 1978) and by activating plasmin (Aoki, 1979). The α2-plasmin inhibitor contains a glutamine substrate. The exact sequence has been identified as NQEQVSPL (SEQ ID NO: 12), with the first glutamine that will be the amino acid for degradation.

It has been shown that two-domain peptides, which contain a Factor XIIIa substrate sequence and a bioactive peptide sequence, can be degraded into fibrin gels and that this bioactive peptide maintains its cellular activity in vitro (Schense, JC, et al . (1999) Bioconj. Chem. 10 : 75-81).

Synthetic matrices

Degradation reactions for the formation of synthetic matrices for the application in which (i) the free radical polymerization between two or more precursors containing unsaturated double bonds is included, as described in Hern et al ., J Biomed Mater . Res. 39 : 266-276 (1998), (ii) the nucleophilic substitution reaction such as, for example, between a precursor that includes an amine group and a precursor that includes a succinimidyl group as set forth in US Pat. No. 5,874,500 of Rhee et al ., (Iii) the condensation and addition reactions and (iv) the Michael-type addition reaction between a strong nucleophile and a conjugated unsaturated group or bond (such as a strong electrophile). Particularly preferred is the reaction between a precursor molecule having a thiol or amine group as the nucleophilic group and the precursor molecules that include acrylate or vinylsulfone groups as electrophilic groups. The thiol group is most preferred as the nucleophilic group. Michael type addition reactions are described in WO 00/44808 by Hubbell et al ., The content thereof is incorporated herein by reference. Michael-type addition reactions allow the in situ degradation of at least a first and a second precursor component under physiological conditions in a self-selective manner, even in the presence of sensitive biological materials. When one of the precursor components has a functional group of at least two, and at least one of the other precursor components has a functional group greater than two, the system will react autoselectively to form a degraded three-dimensional biomaterial.

Preferably, unsaturated groups conjugates or conjugated unsaturated bonds are acrylates, vinyl sulfones, methacrylates, acrylamides, methacrylamides, acrylonitriles, vinyl sulfones, 2- or 4-vinylpyridinium, maleimides, or quinones.

The preferred nucleophilic groups are thiol groups, amino groups, or hydroxyl groups. The thiol groups are practically more reactive than unprocessed amine groups. How was established above, the pH is important in this consideration: the deprotonated thiol is practically more reactive than the protonated thiol. Therefore, the addition reactions that include a conjugated unsaturation, such as, for example, a acrylate or a quinone, with a thiol to convert two components precursors in a matrix will often be carried out better more quickly and self-selectively at a pH of about 8. At the pH of about 8, most of the  thiols of interest are deprived (and in this way are more reagents) and most of the amines of interest are still proton (and thus are less reactive). When it's used a thiol as the first precursor molecule, is quite convenient a conjugate structure that is selective in its reactivity for thiol in relation to amines.

The first and second precursor molecules Suitable include proteins, peptides, polyoxyalkylenes, poly (vinyl alcohol), poly (alcohol ethylene-co-vinyl), poly (acid acrylic), poly (acid ethylene-co-acrylic), poly (ethylxazoline), poly (vinyl pyrrolidone), poly (ethylene-co-vinyl pyrrolidone), poly (maleic acid), poly (acid ethylene-co-maleic), poly (acrylamide), and block copolymers of poly (oxide of ethylene) -co-poly (oxide of propylene) A particularly preferred precursor molecule polyethylene glycol

Polyethylene Glycol (PEG) English) provides a constitutive, convenient block. PEG linear (meaning with two ends) or branched (what means more than two extremes) can be acquired or synthesized easily and then add functional groups to the extreme groups of PEG, to introduce any strong nucleophile, such as by for example, a thiol, or a conjugated structure, such as, for example, an acrylate or a vinyl sulfone. When these components are mixed either with each other or with a corresponding component in an environment slightly basic, a matrix will be formed by the reaction between the first and second precursor component. A PEG component it can be reacted with a component without PEG, and the weight molecular or hydrophilicity of any component can be control to manipulate the mechanical characteristics, the permeability, and the water content of the biomaterial resulting.

These materials in general are useful in medical implants, as will be described in greater detail below. In matrix formation, especially the matrices that are desired to degrade in vivo , peptides, provide a very convenient constitutive block. It is simple to synthesize peptides containing two or more cysteine residues, and this component can then easily serve as the first precursor component with the nucleophilic groups. For example, a peptide with two free cysteine residues will easily form a matrix when mixed with a PEG tri-vinyl sulfone (a PEG having three branches with vinyl sulfones in each of its branches) at a physiological or slightly higher pH (for example , 8 to 9). Gelification can also proceed well at an even higher pH, although with the potential loss of self-selectivity. When the two liquid precursor components are mixed together as they react for a period of a few minutes to form an elastic gel, which consists of a network of PEG chains, which carry the nodes of the network, with the peptides as connecting links . Peptides can be selected as protease substrates, in order to make the network capable of being infiltrated and degraded by cells, as is done in a protein-based network, such as, for example, in a fibrin matrix . Preferably, the sequences in the domains are substrates for enzymes that are involved in cell migration (for example, as substrates for enzymes such as, for example, colagenaza, plasmin, metalloproteinase (MMP) or elastases), although suitable domains will not be limited to this sequence. A particularly useful sequence is a substrate for enzymatic plasmin (see Examples). The degradation characteristics of the gels can be manipulated by changing them from the peptide that serves as the degrading nodes. A gel can be produced that is degradable by collagenaza, but not by plasmin, or by plasmin but not by collagenaza. In addition, it is possible to cause the gel to degrade faster or slower in response to this enzyme, simply by changing the amino acid sequence to alter the K m or K cat, or both, of the enzymatic reaction. In this way a biomaterial that is biomimetic, and that is capable of being remodeled by the normal remodeling characteristics of the cells can be made. For example, this study shows the substrate sites for the important plasmin protease. The gelation of the PEG with the peptide is self-selective.

Optionally, agents can be incorporated biofunctional in the matrix to provide chemical bonding with other species (for example, a tissue surface). To have protease substrates incorporated in the matrix is important when the matrix is formed from PEG vinyl sulfone. Apart from matrices formed from the reaction of PEG acrylates and the PEG thiols, the matrices formed from PEG Vinyl sulfones and PEG thiols do not contain degradable bonds hydrolytically Therefore, the incorporation of substrates from Protease allows the matrix to degrade in the body.

Synthetic matrices are operationally simple to form. Two liquid precursors are mixed, one precursor contains a precursor molecule with nucleophilic groups and the other precursor molecule contains the electrophilic groups. As the solvent can serve physiological saline. Minimum heat is generated by the reaction. Therefore, gelation is carried out in vivo or in vitro , in direct contact with the tissue, without harmful toxicity. In this way, polymers other than PEG can be used, either telehelicidal modified or modified in their side groups.

For most health indications, the internal cell growth rate or cell migration in the matrix in combination with an adapted degradation rate of the matrix is decisive for the total healing response. He potential of hydrolytically nondegradable matrices that will be invaded by cells is primarily a function of the reticular density If the space between the points of branching or nodes is too small in relation to the size of the cells or if the matrix degradation rate, which gives result in the creation of more space within the matrix, it is very  Slow, a very limited healing response will be observed. The healing matrices found in nature, as per example, fibrin matrices, which are formed as a response damage to the body is known to consist of a very separate network which can be invaded very easily by cells. Infiltration is promotes by ligands for cell adhesion that are a part integrated fibrin network.

Matrices made from synthetic hydrophilic precursor molecules, similar to polyethylene glycol, increase in volume in an aqueous environment after the formation of the polymer network. In order to achieve a sufficiently short gelation time (between 3 to 10 minutes at a pH between 7 to 8 and at a temperature in a variation of 36 to 38 ° C) and a quantitative reaction during the in situ formation of the matrix in In the body, the initial concentration of the precursor molecules must be sufficiently high. Under these conditions, the increase in volume could be carried out after the formation of the network, and the necessary initial concentrations could lead to matrices that are too dense for cell infiltration when the matrix is not degradable in an aqueous environment. In this way, increasing the volume of the polymeric network is important to lengthen and increase the space between the branching points.

Regardless of the initial concentration of the precursor molecules, the hydrogels produced from the same synthetic precursor molecules, such as by example, a four-branched PEG vinylsulfone and a peptide with SH groups, will increase in volume to the same content of water in the state of equilibrium. This means that the older you are The initial concentration of the precursor molecules, the greater will be the final volume of the hydrogel when it reaches its state of Balance. If the available space in the body is too much small to allow a sufficient volume increase and in particular if the link formed from the components precursors is not hydrolytically degradable, the index of Cell infiltration and healing response will decrease. How consequently, the optimum between the two must be found contradictory requirements for application in the body. Be have observed good cell infiltration and subsequent responses of healing with a three-dimensional polymer network formed from of the reaction of a branched trifunctional polymer with at least three practically similar ramifications in molecular weight and one second precursor molecule that is at least one molecule bifunctional The equivalent weight index of the groups functional of the first and second precursor molecules is between  0.9 and 1.1. The molecular weights of the ramifications of the first precursor molecule, the molecular weight of the second precursor molecule and the functionality of the points of branching are selected such that the water content of the resulting polymer network is between the% by weight of balance and 92% by weight of the total weight of the polymer network After finishing the water absorption. Preferably, the Water content is between 93 and 95% by weight of the total weight of the Polymeric network and water after completing water absorption. The water absorption term can be reached either when equilibrium concentration is reached or when space available in the biomaterial does not allow an increase in volume additional. Therefore it is preferred that the selection of initial concentrations of the precursor components be so as low as possible This is true for all matrices. which may increase in volume although in particular for those matrices that undergo a degradation supplied by cells that do not contain hydrolytically degradable links in the network polymeric

The balance between gelation time and the low start concentration in particular for gels Hydrolytically non-degradable should be optimized based on the structure of the precursor molecules. In particular, the weight molecular branches of the first precursor molecule, the molecular weight of the second precursor molecule and the degree of branching, that is, the functionality of the points of branching, should be adjusted accordingly. The mechanism of Actual reaction has a minor influence on this interaction.

This first precursor molecule is a polymer of three or four branches with a functional group in the end of each branch and the second precursor molecule is a linear bifunctional molecule, preferably, a peptide that it contains at least two cysteine groups, then the molecular weight of the ramifications of the first precursor molecule and the weight molecular of the second precursor molecule of preference is selected in such a way that the links between the points of branch after network formation have a weight molecular in the range between 10 to 13 kD (under the conditions under which the links are linear, not branched), of preference between 11 and 12 kD. This allows a concentration initial of the sum of the first and second precursor molecules in the range between 8 to 12% by weight, preferably between 9 and 10% by weight of the total weight of the first and second molecules precursor in solution (before the formation of the network). In case of that the degree of branching of the first precursor component increase to eight and the second precursor molecule is still a linear bifunctional molecule, the molecular weight of the bonds between branching points preferably increases to a weight molecular between 18 to 24 kDa. In case the degree of branching of the second precursor molecule increases from linear to a precursor component of three or four branches, the weight molecular, that is, the length of the bonds accordingly will increase. In a preferred embodiment of the present invention, a composition is selected to include as the first precursor molecule a polymer 15 kD, with three branches trifunctional, that is, each branch that has a weight 5 kD molecular and as the second precursor molecule, a bifunctional linear molecule of a molecular weight in the range between 0.5 to 1.5 kD, even more preferably approximately 1 kD Preferably, the first and second precursor component It is a polyethylene glycol.

In a preferred embodiment, the first precursor component includes as functional groups the groups or conjugated unsaturated bonds, a acrylate or a vinyl sulfone and the functional groups of the second precursor molecule include a nucleophilic group, preferably, a thiol or amino group. In another preferred embodiment of the present invention, the first precursor molecule is a 20 kD polymer of four branches (each branch has a molecular weight of 5k Da) which has functional groups at the end of each branching and the second precursor molecule is a molecule bifunctional linear of a molecular weight in the interval between 1 up to 3 kD, preferably between 1.5 and 2 kD. Preferably, the first precursor molecule is a polyethylene glycol that has groups vinyl sulfone and the second precursor molecule is a peptide that It has cysteine groups. In both preferred modes, the initial concentration of the sum of the first and second molecules precursor varies from 8 to 11% by weight, preferably between 9 and 10% by weight of the total weight of the first and second molecules precursor and water (before the formation of the polymer network), of preference between 5 and 8% by weight until reaching a time of gelation less than 10 minutes. These compositions have a gelation time at pH 8.0 and 37 ° C of approximately 3-10 minutes after mixing.

When the matrix contains links hydrolytically degradable, formed, for example, by the Preferred reaction between acrylates and thiols, lattice density regarding cell infiltration is especially important in the beginning, although in an aqueous environment, the links are they will roll and the network will separate, to allow infiltration mobile. With an increase in the degree of total network branching polymeric, the molecular weight of the entanglements, that is, the  Link length should increase.

B. Cell binding sites

Cells interact with their environment through protein-protein interactions. Protein-oligosaccharide and protein-polysaccharide on the cell surface. Extracellular matrix proteins provide a host of bioactive signals to the cell. This dense network is required to support the cells, and many proteins in the matrix have been shown to control cell adhesion, dispersion, migration and differentiation (Carey, Annual Review of Physiology , 53 : 161-177, 1991). Some of the specific proteins that have been shown to be particularly active include laminin, vitronectin, fibronectin, fibrin, fibrinogen and collagen (Lander, Journal of Trends in Neurological Science , 12 : 189-195, 1989). Many studies of laminin have been conducted, and laminin has been shown to play a vital role in the development and regeneration of in vivo media and nerve cells in vitro (Williams, Neurochemical Research , 12 : 851-869, 1987), thus as also in angiogenesis.

Some of the sequences have been identified specific ones that interact directly with cell receptors and they cause either adhesion, dispersion or signal transduction.

It has been shown that laminin, a large multidomain protein (Martin, Annual Review of Cellular Biology , 3 : 57-85, 1987), consists of three chains with various receptor binding domains. These receptor binding domains include the YIGSR sequence (SEQ ID NO: 2) of the B1 laminin chain (Graf, et al ., Cell , 48 : 989-99E, 1987; Kleinman, et al ., Archives of Biochemistry and Biophysics , 272 : 39-45, 1989; and Massia, et al , J: of Biol. Chem. , 268 : 8053-8059, 1993), LRGDN (SEQ ID NO: 3) of the laminin A chain (Ignatius, et al ., J: of Cell Biology , 111 : 709-720, 1990) and PDGSR (SEQ ID NO: 4) of the B1 laminin chain (Kleinman, et al ., 1989). Various other recognition sequences have also been identified. These include IKVAV (SEQ ID NO: 5) of the lamina A chain (Tashiro, et al ., J; of Biol. Chem. , 264 : 16174-16182, 1989) and the RNIAEIIKDI sequence (SEQ ID NO: 6) of the B2 laminin chain (Liesi, et al ., FEBS Letters , 244 : 141-148, 1989). Frequently, receptors that bind to these specific sequences have also been identified. A subset of cellular receptors that has been shown to be responsible for the majority of the union is the integrin superfamily (Rouslahti, E., J. of Clin. Investigation , 87 : 1-5, 1991). Integrins are protein heterodimers consisting of α and β sub-units. Previous work has shown that the RGD tripeptide binds to various β1 and β3 integrins (Hynes, RO, Cell , 69 : 1-25, 1992; Yamada, KM, J; of Biol. Chem. , 266 : 12809- 12812, 1991), IKVAV (SEQ ID NO: 5) binds to a 110 kDa receptor (Tashiro, et al ., J of Biol. Chem. , 264 : 16174-16182, 1989); Luckenbill-Edds, et al ., Cell Tissue Research , 279 : 371-377,1995), YIGSR (SEQ ID NO: 2) binds to a 67 kDa receptor (Graf, et al ., 1987) and DGEA (SEQ ID NO: 7), a collagen sequence, binds to integrin? 2,? 1 (Zutter & Santaro, Amer. J. of Patholody , 137: 113-120, 1990). The receptor for the RNIAEIIKDI sequence (SEQ ID NO: 6) has not been reported.

In an additional preferred embodiment, incorporate peptide sites for cell adhesion in the matrix, namely peptides that bind to receptors to stimulate the adhesion on cell surfaces in biomaterials of the present invention. These peptides to stimulate adhesion They can be selected from the group as described above. In Particularly preferred are the RGD sequence from fibronectin, the YIGSR sequence (SEQ ID NO: 2) from laminin The incorporation of binding sites is a modality particularly preferred with synthetic matrices, but also It can be included with some of the natural matrices. The incorporation can be done, for example, simply by mixing a cell binding peptide containing cysteine with the precursor molecule that includes the conjugated unsaturated group, such such as PEG acrylate, PEG acrylamide or PEG vinyl sulfone a few minutes before mixing with the rest of the component precursor including the nucleophilic group, such as, for example, the precursor component that contains thiol. If the binding site cell does not include a cysteine, this can be synthesized Chemically to include one. During this first step, the peptide to stimulate adhesion will be incorporated at one end of the precursor with multiple functional groups and with unsaturation conjugated; when the remaining multitiol is added to the system, It will form a degraded network. Another important implication of the form in which the networks are prepared here, is the efficiency of incorporation of pendant bioactive ligands such as by example, the signs of adhesion. This step must be quantitative because, for example, unbound ligands (for example, adhesion sites) could inhibit cell interaction with the matrix. As will be described later, the derivation of precursor with these hanging oligopeptides is conducted in a first step in large stoichiometric excess (minimum: 40 times) of multiramified electrophilic precursors on thiols and so both definitely quantitative. Apart from avoiding inhibition unwanted, this achievement is even biologically more significant:  cell behavior is extremely sensitive to small changes in ligand densities and an accurate knowledge of Built-in ligands help design and understand the cell matrix interactions. In summary, the concentration of adhesion sites covalently bound in the matrix influences significantly in the rate of cell infiltration. By For example, for a given hydrogel, a RGD concentration in the matrix with internal growth supports cell, and cell migration in an optimal way. The variation of optimal concentration of adhesion sites similar to GDPR is between 0.04 and 0.05 mM and even more preferably 0.05 mM in particular for a matrix that has an aqueous content between the equilibrium concentration and 92% by weight after the end of the water absorption.

Excellent results of bone healing by maintaining the rate of cell migration and the fasting matrix degradation rate. With respect to matrix design (in particular PTH 1-34 attached covalently), a four-branched polyethylene glycol with a molecular weight of approximately 20,000 Da degraded with a site of protease degradation GCRPQGIWGQDRC (SEQ ID NO: 8) and 0.050 mM GRGDSP (SEQ ID NO: 9) provides growth results particularly good cell internals and defect healing bone. The initial concentration of PEG and 10% inner peptide in weight of the total weight of the molecules and water (before the increase of volume). The gels have a usable consistency and allow that osteoblasts and the precursor cell easily infuse the matrix.

The preferred matrix material is biodegradable by naturally occurring enzymes. The index of degradation can be manipulated by the degree of degradation and the inclusion of protease inhibitors in the matrix.

C. Domains of the degradable substrate

The PTH fusion peptide can be degraded and covalently bound to the matrices through the degradable substrate domain of the PTH fusion peptide. The type of substrate domain depends on the nature of the matrix. For incorporation into fibrin matrices, the transglutaminase substrate domains are particularly preferred. The transglutaminase substrate domain may be a Factor XIIIa substrate domain. This domain of the Factor XIIIa substrate may include GAKDV (SEQ ID NO: 10), KKKK (SEQ ID NO: 11), or NQEQVSPL (SEQ ID NO: 12). The coupling between the PTH and the transglutaminase substrate domain can be performed by chemical synthesis.

The transglutaminase substrate domain can be a substrate for a transglutaminase other than Factor XIIIa. The most preferred Factor XIIIa substrate domain has an amino acid sequence of NQEQVSPL (SEQ ID NO: 12) (hereinafter referred to as "TG"). Other proteins that recognize transglutaminase, such as, for example, fibronectin, could be coupled to the transglutaminase substrate peptide.

TABLE 1 Substrate domains of transglutaminase

one

For the incorporation of PTH in a matrix formed from synthetic precursor components, the peptide fusion are PTH or any other peptide that will be incorporated must be synthesized with at least one additional cysteine group (-SH) preferably in the N term of the PTH as the domain of the degradable substrate. Cysteine can either be attached directly to the PTH or through a binding sequence. Sequence binder may additionally include a sequence of enzymatically degradable amino acids, so that PTH is can cleave the matrix by enzymes practically in the natural form. The free cysteine group reacts with the group unsaturated conjugate of the precursor component in a reaction of Michael type addition. In the case of PTH 1-34, the link with a synthetic matrix for PTH 1-34 se makes it possible by binding an amino acid sequence additional with the term N of PTH 1-34 which contains At least one cysteine. The thiol group of cysteine can react with a conjugated unsaturated bond on the polymer synthetic to form a covalent bond. The possibility that (a) only one cysteine binds to the peptide, in the possibility of that (b) an enzymatically degradable, a degradable sequence of Plasmin binds as a linker between cysteine and the peptide. The GYKNR sequence (SEQ ID NO: 15) between the first domain and the second domain, cysteine, makes the plasmin bond degradable

In this way, the fusion peptides with the PTH, can be further modified to contain a site degradable between the binding site, that is, the second domain (i.e. the domain of the Factor XIIIa or cysteine substrate) and the PTH, that is, the first domain. These sites can be degrade either by non-specific hydrolysis (i.e., a ester bond) or may be substrates for degradation specific enzyme (degradation either proteolytic or polysaccharides). These degradable sites allow treatment by engineering the more specific release of PTH to from matrices similar to fibrin gels. For example, degradation based on enzymatic activity allows the PTH release that will be controlled by a cellular process instead of diffusion of the factor through the gel. The place degradable or binding is cleaved by enzymes released from the cells that invade the matrix.

Degradation sites allow PTH is released with little or no modification with the sequence of primary peptides, which can result in a higher factor activity. In addition, it allows the release of the factor is controlled by specific cellular processes, such as for example, localized proteolysis, instead of diffusion from some porous materials. This allows the factors are released at different speeds within it material depending on the location of the cells within the material. This also reduces the amount of total PTH. necessary, because its release is controlled by processes cell phones. The conservation of PTH and its bioavailability are advantages other than the exploitation of proteolytic activity cell specific regarding the use of devices for diffusion controlled release. In a possible explanation for good healing of a bone defect with united PTH covalently to a matrix, it seems important that PTH be administer locally for an extended period of time (it is say, not just a single pulsed dose) but not in a way keep going. This is accomplished by slow degradation, to through either enzymatic cleavage or hydrolytic cleavage of the matrix. In this way, the molecule is then supplied through a pseudo-pulsed effect that occurs during a sustained time period. When a progenitor cell infiltrates the matrix, it will find a PTH molecule and you can differentiate into a proteoblast. However, if this cell particular does not continue to release the bound PTH from the matrix, it it will effectively become an osteoblast and start the production of bone matrix Finally, the therapeutic effects of the peptide are they locate in the defective region and are subsequently increased.

Enzymes that could be used for Proteolytic degradation are many. The proteolytically sites degradable could include substrates for activators of Colagenaza, plasmin, elastase, stomelysin, or plasminogen. Sample substrates are listed below. P1-P5 denotes positions 1-5 of amino acids towards the Amino term of the protein from the site where the proteolysis P1'-P4 'denote the positions 1-4 amino acids towards the carboxy term of the protein from where proteolysis occurs.

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TABLE 2 Sample substrate sequences for protease

2

In another preferred embodiment, a domain oligo-ester could be inserted between the first and the second domain. This could be done using an oligo-ester such as for example, the oligomers of lactic acid.

The substrate for non-enzymatic degradation it could consist of any link undergoing hydrolysis by a mechanism catalyzed by acid or base. These substrates they can include oligo esters such as, for example, oligomers of lactic or glycolic acid. The degradation rate of these Materials can be controlled through the choice of oligomer

D. PTH

The term "PTH" in the sense in which  used herein, includes the human PTH sequence 1-84 and all modified and allelic versions, truncated, of the PTH that exhibit properties for the formation of bones when they covalently bind to natural matrices or Biodegradable synthetics The preferred truncated versions of the PTH are PTH 1-38, PTH 1-34, PTH 1-31 or PTH 1-25. The most preferred is PTH 1-34. Preferably the PTH is human PTH, although PTH from other sources may be appropriate, such  such as bovine PTH.

Methods for incorporation and / or release of factors bioactive

In a preferred mode for incorporation of a PTH within the matrix, the matrix includes fibrin that is formed from fibrinogen, a source of calcium and thrombin and the PTH fusion peptide will be incorporated into of fibrin during coagulation. The fusion peptide of the PTH is designated as a fusion peptide that includes two domains, one first and one second, a domain, the second, is a substrate for a degrading enzyme such as for example Factor XIIIa. He Factor XIIIa is a transglutaminase that is active during coagulation. This enzyme, formed naturally from the Factor XIII by thrombin cleavage, works to join the fibrin chains with each other via amide bonds, formed between the glutamine side chains and lysine side chains. The  enzyme also works to bind other peptides to fibrin during coagulation, for example, cell binding sites provided also include a Factor XIIIa. Specifically, the  NQEQVSP sequence (SEQ ID NO: 16), has been shown to work as an effective substrate for Factor XIIIa. As described earlier in the present, this is either directly linked to the PTH or may include a degradation site between the PTH (first domain) and the NQEQVSP sequence (SEQ ID NO: 16) (second domain). As such, the PTH fusion peptide can be incorporate into the fibrin during coagulation via a Factor XIIIa substrate.

Design of fusion proteins for incorporation

The PTH fusion peptide that includes a first domain including PTH, a second domain including a substrate domain for a degradation enzyme and optionally a degradation site between the first and the second domain can be incorporated into fibrin gels using Various different schemes. Preferably, the second domain includes a domain of the transglutaminase substrate and even of more preference includes a domain of the Factor substrate XIIIa. Most preferably the domain of the substrate of the Factor XIIIa includes NQEQVSP (SEQ ID NO: 16). When this peptide Fusion with PTH is present during polymerization of the fibrinogen, that is, during the formation of the matrix of fibrin, is incorporated directly into the matrix.

The degradation site between the first and the second domain of the fusion peptide with PTH can be a Enzymatic degradation site as described above. Preferably, the degradation site can be cleaved by an enzyme that selected from the group consisting of plasmin and matrix metalloproteinza. By careful selection of K_ {m} and K cat of this enzymatic degradation site, the degradation It could be controlled to present either before or after the protein matrix and / or when using similar or different enzymes to degrade the matrix, with the degradation site placement  It is prepared for each type of protein and application. This fusion peptide with PTH could be directly degraded in the fibrin matrix as described above. However, the incorporation of an enzyme degradation site alters the PTH release during proteolysis. When proteases cell derivatives reach the sequestered fusion peptide, can cleave the engineered protein and the site of newly formed degradation. Degradation products resulting could include the released PTH, which could now be almost free of any fusion sequences subjected to engineering, as well as any degraded fibrin.

II. Method of use

The matrices can be used for repair, regeneration, or remodeling of tissues, and / or the PTH release, before or at the time of implantation. In some cases it will be convenient to induce site degradation of administration to conform the matrix to the tissue at the site of implantation. In other cases, it will be convenient to prepare the matrix before implantation.

Cells can also be added to the matrix before or at the time of implantation, or even after of implantation, either at the time or after the degradation of the polymer to form the matrix. This can be perform in addition or instead of matrix degradation to produce interstitial separation designed to stimulate cell proliferation or internal growth.

Although in most cases it will be convenient to implant the matrix to stimulate growth or cell proliferation, in some cases bioactive factors are will use to inhibit the rate of cell proliferation. A specific application is to inhibit the formation of adhesions after of surgery

III. Application methods

In the preferred embodiment, the material gels in situ inside or on the body. In another embodiment, the matrix can be formed outside the body and then applied in the preformed conformation. The matrix material can be produced from synthetic or natural precursor components. Regardless of the type of precursor component used, the precursor components must be separated prior to application of the mixture to the body to avoid combination or contact with each other under conditions that allow polymerization or gelation of the components. To make the contact before administration, a reagent kit can be used that separates the compositions from each other. At the time of mixing at conditions that allow polymerization, the compositions form a three-dimensional network complemented with the bioactive factor. Depending on the precursor components and their concentrations, gelation can occur almost instantaneously after mixing. This rapid gelation makes injection almost impossible, that is, pressure extraction of the gelled material through the injection needle.

In one embodiment, the matrix is formed from of fibrinogen. Fibrinogen, through a cascade of several reactions gels to form a matrix, when put in contact with thrombin and a source of calcium at temperature and pH adequate. The three components, fibrinogen, thrombin and the source of calcium, should be stored separately. However, always that at least one of the three components be kept separate, the two other components can be combined before the administration.

In a first mode the fibrinogen (which may additionally contain aprotinin for increase stability) in a buffer solution at a pH physiological (in the range of pH 6.5 to 8.0, preferably 7.0 to 7.5) and stored separately from a solution of thrombin in a calcium chloride buffer (for example, to a concentration that varies from 40 to 50 mM). The solution Fibrinogen buffer may be a solution histidine buffer at a preferred concentration of 50 mM additionally including NaCl at a preferred concentration of 150 mM or buffered TRIS saline solution (preferably at a 33 mM concentration).

In a preferred embodiment, a reagent kit, which contains a fusion protein, Fibrinogen, thrombin and a source of calcium. Optionally, the reagent equipment may contain a degrading enzyme, such as for example Factor XIIIa. The fusion protein contains a bioactive factor, a substrate domain for an enzyme degradant and a degradation site between the substrate domain and the bioactive factor. The fusion protein may be present. in the solution of either fibrinogen or thrombin. In one mode preferred, the fibrinogen solution contains the protein of fusion.

Preferred solutions are mixed with a two-way syringe device, in which the mixed by pressure extraction the contents of both syringes to through a mixing chamber and / or needle and / or mixer static

In a preferred embodiment, both fibrinogen As thrombin are stored separately in lyophilized form. Either one can contain the fusion protein. Prior to used, Tris or histidine buffer is added to the fibrinogen, the buffer may additionally contain aprotinin Lyophilized thrombin dissolves in the solution of calcium chloride. Subsequently, fibrinogen solutions and thrombin are placed in separate vial and syringe containers and are mixed by a two way connection device, such such as a two way syringe. Optionally, the vial / syringe containers are divided into two parts thus having two cameras separated by an adjustable division that is perpendicular to the wall of the syringe body. One of the Chambers contains fibrinogen or lyophilized thrombin, while the other chamber contains a buffer solution adequate. When the plunger is pressed down, the division is move and release the damper inside the chamber of fibrinogen to dissolve fibrinogen. Once they dissolve both fibrinogen and thrombin, both syringe bodies divided into two parts join a connection device of two ways and the contents are mixed when removed by pressure to through the injection needle attached to the connection device. Optionally, the connection device contains a mixer static to improve the mixing of the contents.

In a preferred embodiment, the fibrinogen is dilute eight times and thrombin is diluted 20 times before mixed. This proportion results in a time of gelation of about one minute.

In another preferred embodiment, the matrix is formed a, from synthetic precursor components capable of experience an addition reaction Michael. Because the nucleophilic precursor component (multitiol) only reacts with the multiaceptor component (the unsaturated group conjugate) at basic pH, the three components that must be stored separately before mixing are: the base, the component nucleophilic and the multiaceptor component. Both the multiaceptor as the multitiol component are stored as a solution in shock absorbers. The two compositions may include the site of cell binding and additionally the bioactive molecule. Thus, the first composition of the system for example can include the solutions of the nucleophilic component and the second composition of the System can include the multi-receiver component solution. Either or both compositions may include the base. In other modality, multiaceptor and multitiol can be included as solution in the first composition and the second composition can include the base. The connection and mixing are presented in the same form as described above for fibrinogen. The body of the syringe divided into two parts is also Suitable for synthetic precursor components. Instead of fibrinogen and thrombin, multiaceptor and components Multitiol are stored in powdered form in one of the chambers and The other chamber contains the basic shock absorber.

The following examples are included for demonstrate preferred embodiments of the invention. While the compositions and methods have been described in the terms and Preferred modalities, will be apparent to someone with experience in the art that variations can be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the spirit concept and scope of the invention.

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Example 1 Matrices containing covalently bound TGPTH TGPTH synthesis

PTH peptide 1-34-mer showing activity similar with the whole protein, and proteins of this length are they can synthesize by synthesis methods for peptides in standard solid state.

All peptides were synthesized on resin solid using an automatic peptide synthesizer using the standard 9-fluorenylmethyloxycarbonyl chemistry. The peptides were purified by c18 chromatography and were analyzed using reverse phase chromatography via HPLC to determine purity, as well as mass spectroscopy (MALDI) to identify the molecular weight of each product. Using this method, the following peptide was synthesized, (referred to herein as "TGPTH"):

100

Results in vivo

The activity of TGPTH to improve bone regeneration was tested on a TISSUCOL® matrix in a hole-drilled defect in a sheep. Holes of 8 mm and 12 mm depth were created in the femur and proximal and distal humerus of a sheep. These holes were filled with an in situ polymerizing fibrin gel. The defects were left empty, filled with TISSUCOL® or TGPTH was added to the TISSUCOL® fibrin at 400 µg / mL before polymerization. In each example in which TISSUCOL® was used, it was diluted four times from the standard concentration available, leading to a fibrinogen concentration of 12.5 mg / mL.

The defects were allowed to cure for eight weeks After this healing period, the animals will they sacrificed, and the bone samples were removed and analyzed by microcomputed topography (µCT). Then it was determined the percentage of the defective volume filled with bone tissue calcified When the defects were left empty, there was no formation of calcified tissue inside the matrix of fibrin. When only one fibrin gel was added, there was virtually no bone healing. However, with the addition of 400 g / mL of TGPTH, the cure level increased dramatically, with the defect filled with 35% bone calcified

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Example 2 Healing response with modified PTH 1-34 attached to a fibrin matrix materials

The modified version of PTH_ {1-34} that can be incorporated into an array of fibrin has been tested for the healing response in the cranial defect of critical size in rats.

A fibrin gel was made from fibrin sealant precursor components of the equipment for TISSUCOL® reagents (Baxter AG, CH-8604 Volketswil / ZH). The fibrinogen was diluted in Tris solution 0.03M buffered sterile (TBS, pH 7.4) to form a solution of approximately 8 mg / mL and the thrombin was diluted in solution of 50 mM sterile CaCl2 to form a 2 U / mL solution. The Final fibrinogen concentration was the TISSUCOL® formulation original 1: 8 (approximately 100 mg / mL) and the concentration of Original TISSUCOL® thrombin 1: 160 (approximately 500 IE / mL). Then a predetermined amount of TG-pl-PTH_ {1-34} or TGPTH_ 1-34 to thrombin, and mixed to form a homogeneous concentration.

To form the fibrin gene, the diluted precursors were mixed together when fibrinogen was injected into a tube containing thrombin. In the case of the defect perforated in a sheep (as will be described later), this mixture was then immediately injected into a perforated effect created in a cancerous bone of the sheep, where a fibrin gel formed within 1-5 minutes. In the first series of animal experiments, the efficacy of a fusion protein containing PTH_ 1-34 as the bioactive factor was tested (NQEQVSPLYKNRSVSEIQLMHNLGKHLNS
MERVEWLRKKLQDVHNF, SEQ ID NO: 18) in the healing of cortical bones in a small animal model. The YKNR sequence (SEQ ID NO: 19) makes the plasmin bond degradable (`` TG-pl-PTH_ {1-34}). TG-pl-PTH 1-34 is produced by chemical synthesis. Purification was carried out through reverse phase HPLC (Column C18) by using a TFA as the counterion that resulted in a final product that was a TFA salt. The purity of TG-pl-PTH 1-34 was determined to be 95%.

The healing response was explored in times of healing both short (3 weeks) and long (7 weeks) for determine if an improvement in healing could be observed.

Critical size cranial defect in rats

The rats were anesthetized and the spindle was exposed cranial. The periosteum on the outer surface of the skull is retracted in such a way that it could not play a role in the healing process, and an individual round defect of 8 was created mm This defect size was selected as it had been previously determined that defects of 8 mm or greater are not they heal spontaneously by themselves, and are size defects critical. The defect was then endowed with a fibrin matrix preformed and the animal was allowed to cure for 3 and 7 weeks. The defective region was then explanted and analyzed via radiology as well as histology.

Results

When it was studied TG-pl-PTH_ {1-34} at the time of 3 weeks, the level of healing was very similar to observed with a fibrin matrix alone. The time of 3 weeks is selected as an early time according to other powerful morphogens, including rhBMP-2, showed an important effect Healing in 3 weeks. On the contrary, the healing effect for TG-pl-PTH_ {1-34}, It could not be observed at this early point of time. But nevertheless, when the longest time (7 weeks) was analyzed, a improvement that depends on a moderate dose in healing in the Critical size cranial defect in rats with the addition of PTH_ {1-34} modified to fibrin matrices. The Results are shown in Table 3. When the high dose was used of modified PTH_ {1-34}, the response to the Healing increased by 65%.

TABLE 3 Cure response in cranial defect in rats with modified PTH

3

These results show that when PTH_ 1-34 was incorporated into a fibrin matrix, this maintained some activity as evidenced by the modest increase in bone formation.

Bone piercing defect in sheep

TG-pl-PTH_ 1-34 was also tested in a model with a large bone defect to test the effect of this hormone on bone healing. In the model with a defect in perforation in sheep, cylindrical perforation defects of 8 mm were placed that were approximately 15 mm deep in both the distal and the proximal region of the femur and humerus bones. Because the defect was placed in the epiphysis of long bones, the defect was surrounded by trabecular bone with a thin layer of cortical bone at the edge of the defect. The defects were then filled with an in situ polymerizing fibrin (approximately 750 µL) that contained various doses of TG-pl-PTH 1-34, or TGPTH 1-34. The animals were allowed to cure for eight weeks and then were sacrificed. The defect was analyzed with µCT and histology.

For this series of experiments, they were tested Three types of compositions. First, it was tested TG-pl-PTH_ {1-34} over a large variation of concentration. Second, it employed another modified PTH_ {1-34}, the TGPTH_ {1-34} (NQEQVSPLSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF; SEQ ID NO: 17) that only had a transglutaminase sequence in the term amino, and a degradation site. In this way, the TGPTH_ {1-34} could only be released by degradation of the fibrin matrix itself. TGPTH_ 1-34 was produced and purified in a manner similar to TG-pl-PTH_ {1-34}. The purity was determined to be 95%. TGPTH_ 1-34 was tested at various concentrations that were similar to the concentrations of TG-pl-PTH_ {1-34} To compare the effectiveness. Finally, the matrices were produced in presence of granular material, with either TGPTH_ {1-34} or TG-pl-PTH_ {1-34}. The granular material was a mixture of tricalcium phosphate standard / hydroxyapatite that was incorporated into the matrix during gelation The effect of adding these granules on the the effectiveness of PTH_ {1-34}. As a control, it tested unmodified fibrin.

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Results

When any of the molecules of Modified PTH_ {1-34} was placed in defects of long bone, a significant improvement in responses was observed of cure with respect to the use of fibrin matrices (control) alone The use of fibrin alone resulted in poor healing, where only 20% of the original defect was filled with newly formed bone.

It was tested TG-pl-PTH_ {1-34}, in a concentration series of 20-1000 µg / mL. For each dose tested, a significant increase in the healing response. For example, when 100 were used \ mug / mL of TG-pl-PTH_ {1-34}, The cure rate was increased by almost 60%.

In a second series of experiments, it was tested TGPTH_ {1-34}. The use of TGPTH_ 1-34 also increased bone healing. By For example, the use of 400 µg / mL improved the healing response to 40%, and 1000 µg / mL increased bone healing to 65%. This way, adding any sequence of Modified PTH_ {1-34} resulted in a healing response more important than control.

Finally, when any molecule of Modified PTH_ {1-34} joined the matrix and was used a mixture of granule / matrix, the effectiveness of the PTH_ {1-34}. This was proven so much for TG-pl-PTH_ {1-34} (see Table 4) as for TGPTH_ {1-34} (see Table 5).

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TABLE 4 Healing of a defect in sheep drilling with TG-pl-PTH_ {1-34} incorporated into a fibrin matrix; healing time of 8 weeks

4

TABLE 5 Healing of a defect in sheep drilling with PTH 1-34 bound to a fibrin matrix; 8 week cure time

5

Histological evaluation showed high infiltration in the original defect of the progenitor cells fusiform and osteoblasts supported on a matrix extracellular Active osteoids with large osteoblasts rounded were common, and dosage signs were observed  indochondral (chondrocytes). For eight weeks, they could Find osteoclasts and healthy signs of remodeling. Though unlike the results obtained from the exhibition continued to systemic PTH, no response was observed manifests from osteoclasts and the formation of new bones was significantly greater than that of absorption in the area of defect and around it. No response detected inflammatory foreign body (i.e. there were no giant cells and only a slight presence of monocytes). They were still granules present in samples with mineral particles added.

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Example 3 Preparation of the precursor components for matrices synthetic Preparation of PEG-vinyl sulfones

Commercially available branched PEGs (PEG of 4 branches, molecular weight of 14,800, PEG of 4 Branches, molecular weight of 10,000 and PEG of 8 branches, molecular weight of 20,000; Shearwater Polimers, Huntsville, AL, USA) they were functional groups in the term OH.

PEG vinyl sulfones were produced under argon atmosphere by reacting a solution of dichloromethane of the precursor polymers (pre-dried on molecular sieves) with NaH and then, after evolution with hydrogen, with divinylsulfone (molar proportions: OH 1: NaH 5: divinylsulfone 50). The reaction was carried out at temperature ambient for 3 days under argon with constant agitation. After of neutralization of the reaction solution with acetic acid concentrated, the solution was filtered through paper until It was clear. The derivative polymer was isolated by precipitation in ice-cold diethyl ether. The product was dissolved again in dioloromethane and precipitated again in diethyl ether (with rigorous washing) twice to remove all excess divinylsulfone. Finally, the product was dried under vacuum. The shunt was confirmed with 1 H NMR. The product showed peaks of Characteristic vinyl sulfone at 6.21 ppm (two hydrogen) and 6.97 ppm (a hydrogen). It was found that the degree of conversion of the group Final was 100%.

Preparation of PEG-acrylates

PEG acrylates were produced under an argon atmosphere by reacting an azeotropically toluene solution of the precursor polymers with acryloyl chloride, in the presence of triethylamine (molar proportions: OH 1: acryloyl chloride 2: triethylamine 2.2). The reaction continued with stirring overnight in the dark at room temperature. The resulting pale yellow solution was filtered through a bed of neutral alumina; After evaporation of the solvent, the reaction product was dissolved in dichloromethane, washed with water, dried over sodium sulfate and precipitated in cold diethyl ether. Yield: 88%; OH to acrylate conversion: 100% (from 1 H-NMR analysis) 1 H-NMR (CDCl 3): 3.6 (341H (14800 4 branches: theoretical 337H), 230 ( 10000 4 branches theoretical 227H), or 210H (20,000 8 branches: theoretical 227H), PEG chain protons), 4.3 (t, 2H, -CH2 -C H2 -O-CO-CH = CH2), 5.8 (dd, 1H, CH2 = C H -COO-), 6.1 and 6.4 (dd, 1H, C H2 = CH-COO-) ppm. FT-IR (film on an ATR plate): 2990-2790 (\ nu CH), 1724 (\ nu C = O), 1460 (\ nu_ {s} CH_ {2}), 1344, 1281, 1242, 1097 ( nuCOC), 952, 842 (cmCOC) cm -1.

Peptide synthesis

All peptides were synthesized on resin solid using an automatic peptide synthesizer (9050 Pep Plus Synthesizer, Millipore, Framingham, USA) with chemical 9-fluorenylmethyloxycarbonyl standard. The hydrophobic scrubbers and cleaved protecting groups will removed by precipitation of the peptide in cold diethyl ether and dissolution in deionized water. After lyophilization, the peptides were re-dissolved in saline Tris-buffered 0.03 M (TBS, pH 7.0) and se purified using HPLC (Waters; Milford, USA) on a size exclusion column with TBS, pH 7.0 as the shock absorber in progress.

Formation of the matrix by addition reactions conjugated

MMP-sensitive gels were formed by the conjugate addition with a peptide-linked nucleophile and a conjugated unsaturation linked by PEG that allows the migration of proteolytic cells. The synthesis of the gels is carried out completely through a Michael-type addition reaction of thiol-PEG on PEG with a vinyl sulfone functional group. In a first step, adhesion peptides (for example, the Ac-GCGYG RGDSPG -NH2 (SEQ ID NO: 20) peptide) were pendently bound to a multiramified PEG-vinylsulfone and then this precursor was degraded with a dithiol-containing peptide (for example, the MMP substrate Ac-GCRDG PQGIAGF DRCG-NH2 (SEQ ID NO: 21)). In a typical gel preparation for three-dimensional in vitro studies, 4-branched PEG-vinylsulfone (15,000 molecular weight) was dissolved in a TEOA buffer (0.3M, pH 8.0) to provide a 10% solution (w / w). In order to make the gels adhesive with cells, the dissolved peptide Ac-GCGYG RGDSP G- NH2 (SEQ ID NO: 20) (same buffer) was added to this solution. The adhesion peptide was allowed to react for 30 minutes at 37 ° C. After this, the Ac-GCRD GPQGIWGQ DRCG-NH2 degrading peptide (SEQ ID NO: 21) was mixed with the above solution and the gels were synthesized. The gelation occurred in a few minutes, however, the degradation reaction was carried out for one hour at 37 ° C to ensure the complete reaction.

Non- MMP sensitive gels were formed by the addition of conjugates with a PEG-linked nucleophile and a conjugated unsaturation linked with PEG that allows non-proteolytic cell migration.

The synthesis of gels was also carried out completely through the Michael-type addition reaction of thiol-PEG on PEG with a vinyl sulfone functional group. In a first step, the adhesion peptides were attached in a pendant manner (for example, the Ac-GCGYG RGDSP G-NH2 peptide (SEQ ID NO: 20)) to a multiramified PEG vinylsulfone and then this precursor was degraded with a PEG-dithiol (pm 3.4 kD). In a typical gel preparation for three-dimensional in vitro studies, the 4-branched PEG vinylsulfone (molecular weight 15000) was dissolved in a TEOA buffer (0. 3M, pH 8.0) to provide a 10% solution (w / w). In order to make the cell adhesive gels, the dissolved peptide Ac-GCGYG RGDSP G- NH2 (SEQ ID NO: 20) (in the same buffer) was added to this solution. The adhesion peptide was allowed to react for 30 minutes at 37 ° C. After this, the PEG-dithiol precursor was mixed with the above solution and the gels were synthesized. The gelation occurred in a few minutes, however, the degradation reaction was carried out for one hour at 37 ° C to ensure the complete reaction.

Formation of the matrix by condensation reactions

MMP sensitive gels were formed by condensation reactions with an X-binder peptide containing multiple amines and an electrophilically PEG active that allows proteolytic cell migration.

MMP-sensitive hydrogels were also created by conducting a condensation reaction between the MMP-sensitive oligopeptide containing two MMP and three Lys substrates (Ac-G K GPQGIAGQ K GPQGIAGQ K G- NH_ {2} (SEQ ID NO: 22) and a commercially available difunctional double ester PEG-N-hydroxysuccinimide (Shearwater polimers) (NHS-HBS-CM-PEG-CM-HBA-NHS) In a first step, one of the adhesion peptides (e.g., the Ac peptide -GCGYG RGDSP G- NH 2 {}) (SEQ ID NO: 20) was reacted with a small fraction of NHS-HBS-CM-PEG-CM-HBA-NHS and then this precursor was degraded to a network by mixing with Ac-G KGPQGIAGQKG PQGIAGQK G- NH 2} {(SEQ ID NO: 22) peptide. carrying three \ epsilon amines (and a primary amine) in a typical preparation of gel in dimensional vitro studies, the two components dissolved in 10 mM PBS at pH 7.4 to provide a 10% solution (w / w) and the hydrogels formed in less than an hour.

In contrast to the hydrogels present formed by the Michael type reaction, the desired self-selectivity in this procedure, because the amines present in biological materials similar to cells or tissues will also react with activated double esters difunctional This is also true for other PEGs that carry electrophilic functional groups such as, for example, PEG-Oxycarbonylimidazole (CDI-PEG), or PEG nitrophenyl carbonate.

Non- MMP sensitive hydrogels were formed by condensation reactions with a PEG-amine degradant and an electrophilically active PEG that allows non-proteolytic cell migration.

Hydrogels were also formed by conducting a condensation reaction between commercially available branched PEG-amines (Jeffamines) and the same difunctional double ester PEG-N-hydroxysuccinimide (NHS-HBS-CM-PEG-CM-HBA-NHS). In a first step, adhesion peptides (for example, the Ac-GCGYG RGDSP peptide
G- NH2 ) (SEQ ID NO: 20) were reacted with a small fraction of NHS-HBS-CM-pEG-CM-HBA-NHS and then this precursor degraded to a network when mixed with the PEG amine multiramified. In a typical gel preparation for three-dimensional in vitro studies, the two components were dissolved in 10 mM PBS at pH 7.4 to provide a 10% solution (w / w) and hydrogels formed in less than an hour.

Again, in contrast to hydrogels present formed by the Michael type reaction, I don't know guaranteed the desired self-selectivity in this procedure, due to to the amines present in biological materials similar to cells or tissues will also not react with double esters difunctional activated. This is also true for other PEGs. that carry electrophilic functional groups such as by example, PEG-oxycarbonylimidazole (CDI-PEG), or PEG nitrophenyl PEG carbonate.

Example 4 Bone regeneration with enzymatically degradable matrices synthetic

Two starting concentrations were used different from enzymatic degradable gels. In each of these, the concentration of GDPR and the active factor (CplPTH at 100 µg / mL) remained constant. The polymer network was formed to from a PEG with a functional group of four branches with four final vinyl sulfone groups with a molecular weight of 20 kD (molecular weight of each of the 5kD branches) and peptide of dithiol of the following sequence Gly-Cys-Arg-Asp- (Gly-Pro-Gln-Gly-Ile-Trp-Gly-Gln) -Asp-Arg-Cys-Gly (SEQ ID NO: 21). The two precursor components dissolved in triethanolamine 0.3 M. The initial concentration of PEG was varied with functional group (first precursor molecule) and the peptide dithiol (second precursor molecule). In one case the concentration it was 12.6% by weight of the total weight of the composition (first and second precursor component + triethanolamine solution). 12.6%  by weight corresponds to a 10% solution by weight when Calculates on the basis of only the first precursor component (100 mg / mL of the first precursor molecule). The second baseline concentration was 9.5% by weight of the total weight of the composition (first and second precursor component + solution of triethanolamine) corresponding to 7.5% by weight based on only the first precursor molecule (75 mg / mL of the first precursor molecule) of the total weight. This had the consequence of that the amount of the dithiol peptide was changed such that maintained the molar ratio between vinyl sulfones and thiols

The gel that started from a initial concentration of 12.6% by weight increased in volume to a 8.9% concentration by weight of the total weight of the polymer network more water, in this way the matrix had an aqueous content of 91.1. The gel that started from an initial concentration of 9.5% in weight increased in volume to a final concentration of 7.4% in weight of the total weight of the polymer network plus water, in this way It had an aqueous content of 92.6.

In order to explore the effect of this change, these materials were tested on the defect of perforation in a sheep. Here, a 750 µL defect was placed in the cancerous bone of the sheep's femur and humerus diaphysis and filled with an in situ gelatinizing enzyme gel. The following amount of calcified tissue, determined via µCT, was obtained with each group at N = 2.

       \ dotable {\ tabskip \ tabcolsep \ hfil # \ hfil \ + \ hfil # \ hfil \ + \ hfil # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
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By making gels less dense and easier for cell penetration, the resulting healing response with the addition of an active factor it was stronger. The effect of having final solid concentrations less than 8.5% by weight is Obvious from these results.

So clearly, the design of the matrix is crucial to allow healing in wound defects. Each of these hydrogels was composed of large chains of polyethylene glycol, linked at the end to create a matrix. Without  However, the details of how they were linked, via sites of enzymatic degradation, density of linkers and other various variables were crucial to allow a response from functional healing These differences were observed very clearly in the model of defect of perforation in the sheep.

Example 5 Bone formation with hydrolytically degradable matrices synthetic

A fusion peptide was tested with PTH 1-34 in a synthetic gel as well as in the drilling defect model in a sheep exactly as it is described for fibrin matrices. A hydrogel network was created by mixing 4-branched polyethylene glycol together acrylated, PM 15,000 (Peg 4 * 15 Acr) with a polyethylene glycol dithiol Linear PM 3400. Through a Michael-type reaction, when the two components were mixed in a shock absorber 0.3 M triethanolamine at pH 8.0, the resulting thiolates that are formed at this pH then reacted with unsaturation acrylate conjugate to create a covalent bond. When mixing together the multifunctional precursors, so that the combined multifunctionality was greater than or equal to five, a hydrogel In addition, biactive factors can be added to the matrix a through an identical reaction scheme. In this case, the bioactive factors, including reasons for cell adhesion or morphogenic or mitogenic factors could bind to the matrix by adding a cysteine, the thiol that contains amino acids, to the sequence. Here, they had to add a cysteine to the cell adhesion sequence, RGD, and more specifically, RGDSP (SEQ ID NO: 23), as well as the sequence of PTH_ {1-34} and both joined the matrix via the acrylates in the degradant. Subsequently, these newly hydrogels formed, then they had many esters close to a thiol, what which has been shown to be hydrolytically unstable. This instability allow gels to degrade slowly and be replaced by newly formed tissue.

These particular, degradable gels hydrolytically with RGD and PTH covalently bound to the matrix, it they tested on the model of drilling defect in a sheep to test the ability of the C-PTH_ {1-34} attached to the matrix to improve bone development. In order to determine the amount of improvement, 0, 100 and 400 µg / mL were added to the matrix of the fusion peptide with PTH 1-34. When performed this, an increase in bone formation was observed with the addition of PTH_ {1-34}. In each test the healing response at the same time of eight weeks. This is compared with the defects that were left empty. When the  synthetic hydrolytically degradable matrix without the peptide of fusion with PTH, the cure response was measured in approximately 40% This means that 40% of the volume of Original defect was filled with freshly formed bone tissue. After,  when 400 µg / mL of the fusion peptide was used with the PTH_ {1-34} modified, the healing response increased to approximately 60%. In comparison, when the defects they were left empty, approximately 10% was filled with tissue calcified These data are shown in the following Table 6.

TABLE 6 Cure response with synthetic matrices with and no modified PTH

6

Compared to an empty defect, the addition of the hydrolytically degradable peg gel only had a great effect about bone healing, increasing the amount of tissue calcified by 300%. When PTH 1-34 joined to this matrix, healing increased even more with the level that was up to 50% higher than when the matrix was used alone and in 500% higher than the level of healing obtained when the Default was left empty.

<110> Eidgenossiche Technische Hochschule Zurich, University of Zurich, and Jeffrey A. Hubbell

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         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> MOD_RES

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (1)

         \ vskip0.400000 \ baselineskip
      

<223> ACETYLATION

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> MOD_RES

         \ vskip0.400000 \ baselineskip
      

<222> (16) .. (16)

         \ vskip0.400000 \ baselineskip
      

<223> AMIDATION

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 21

         \ vskip1.000000 \ baselineskip
      

\ sa {Gly Cys Arg Asp Gly Pro Gln Gly Ile Trp Gly Gln Asp Arg Cys Gly}

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 22

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> MOD_RES

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (1)

         \ vskip0.400000 \ baselineskip
      

<223> ACETYLATION

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> MOD_RES

         \ vskip0.400000 \ baselineskip
      

<222> (21) .. (21)

         \ vskip0.400000 \ baselineskip
      

<223> AMIDATION

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 22

9

         \ vskip0.400000 \ baselineskip
      

<210> 23

         \ vskip0.400000 \ baselineskip
      

<211> 5

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 23

         \ vskip1.000000 \ baselineskip
      

\ sa {Arg Gly Asp Ser Pro}

         \ vskip1.000000 \ baselineskip
      

Claims (19)

1. A fusion peptide, characterized in that it comprises: a first domain comprising PTH and a second domain comprising a domain of covalently degradable substrate. 2. The fusion peptide according to claim 1, further characterized in that it comprises a degradation site between the first and second domain. 3. The fusion peptide according to claim 1, characterized in that the PTH is selected from the group consisting of PTH 1-84, PTH 1-28, PTH 1-34, PTH 1-31 and PTH 1-25. 4. The fusion peptide according to claim 3, characterized in that the PTH is PTH 1-34. 5. The fusion peptide according to claim 1, characterized in that the second domain comprises a transglutaminase substrate domain. 6. The fusion peptide according to claim 5, characterized in that the second domain comprises a Factor XIIIa substrate domain. 7. The fusion peptide according to claim 6, characterized in that the domain of the Factor XIIIa substrate comprises SEQ ID NO: 12. 8. The fusion peptide according to claim 1, characterized in that the second domain comprises at least one cysteine. 9. The fusion peptide according to claim 2, characterized in that the degradation site is an enzymatic or hydrolytic degradation site. 10. The fusion peptide according to claim 8, characterized in that the degradation site is an enzyme degradation site, which is cleaved by an enzyme selected from the group consisting of plasmin and matrix metalloproteinase. 11. A reagent kit characterized in that it comprises the fusion peptide according to claims 1-10. 12. The reagent kit according to claim 11, further characterized in that it comprises fibrinogen, thrombin and a calcium source. 13. The reagent kit according to claim 11, characterized in that the reagent kit also comprises a degrading enzyme. 14. A matrix suitable for internal cell growth or growth, comprising the fusion peptide according to claims 1-10, characterized in that the fusion peptide is covalently linked to the matrix. 15. The matrix according to claim 14, characterized in that the matrix is fibrin. 16. The matrix according to claim 14, characterized in that the matrix is formed by a Michael-type addition reaction between a first precursor molecule comprising n nucleophilic groups and a second precursor molecule comprising m electrophilic groups, wherein n and m are at minus two and the sum n + m is at least five. 17. The matrix according to claim 16, characterized in that the electrophilic groups are conjugated unsaturated groups and the nucleophilic groups are selected from the group consisting of thiols and amines. 18. The matrix according to claim 14, characterized in that the matrix comprises polyethylene glycol. 19. A method for developing a matrix characterized in that it comprises: provide at least one capable matrix material of forming a degraded matrix, where the matrix material is select from the group consisting of proteins and materials synthetic; add the fusion peptide according to claims 1-10 to the parent material, and degrade the matrix material, so that the fusion peptide binds to the matrix through the second domain.